Molecular modelling of coat protein of the Groundnut bud necrosis tospovirus and its binding with Squalene as an antiviral agent: In vitro and in silico docking investigations.

Bud blight disease caused by groundnut bud necrosis virus (GBNV) is a serious constraint in the cultivation of agricultural crops such as legumes, tomato, chilies, potato, cotton etc. Owing to the significant damage caused by GBNV, an attempt was made to identify suitable organic antiviral agents through molecular modelling of the nucleocapsid Coat Protein of GBNV; molecular docking and molecular dynamics that disclosed the interaction of the ligands viz., Squalene and Ganoderic acid-A with coat protein of GBNV. Invitro inhibitory effect of Squalene and Ganoderic acid-A was examined in comparison with different concentrations, against GBNV in cowpea plants under glasshouse condition. The different concentrations of Squalene (50, 100, 150, 250 and 500 ppm) tested in vitro resulted in reduction of lesion numbers (1.69 cm2) as well as reduced virus titre in co-inoculation spray. The present study suggests the antiviral activity of Squalene by effectively fitting into binding site of coat protein of GBNV with favourable hydrophilic as well as strong hydrophobic interactions thereby challenging and blocking the binding of viral replication RNA with coat protein and propagation. The present organic antiviral molecules will be helpful in development of suitable eco-friendly formulations to mitigate GBNV infection disease in plants.

Identification of a Common Epitope in Nucleocapsid Proteins of Euro-America Orthotospoviruses and Its Application for Tagging Proteins.

The NSs protein and the nucleocapsid protein (NP) of orthotospoviruses are the major targets for serological detection and diagnosis. A common epitope of KFTMHNQIF in the NSs proteins of Asia orthotospoviruses has been applied as an epitope tag (nss-tag) for monitoring recombinant proteins. In this study, a monoclonal antibody TNP MAb against the tomato spotted wilt virus (TSWV) NP that reacts with TSWV-serogroup members of Euro-America orthotospoviruses was produced. By truncation and deletion analyses of TSWV NP, the common epitope of KGKEYA was identified and designated as the np sequence. The np sequence was successfully utilized as an epitope tag (np-tag) to monitor various proteins, including the green fluorescence protein, the coat protein of the zucchini yellow mosaic virus, and the dust mite chimeric allergen Dp25, in a bacterial expression system. The np-tag was also applied to investigate the protein-protein interaction in immunoprecipitation. In addition, when the np-tag and the nss-tag were simultaneously attached at different termini of the expressed recombinant proteins, they reacted with the corresponding MAbs with high sensitivity. Here, we demonstrated that the np sequence and TNP MAb can be effectively applied for tagging and detecting proteins and can be coupled with the nss-tag to form a novel epitope-tagging system for investigating protein-protein interactions.

A Conserved Helix in the C-Terminal Region of Watermelon Silver Mottle Virus Nonstructural Protein S Is Imperative For Protein Stability Affecting Self-Interaction, RNA Silencing Suppression, and Pathogenicity.

In orthotospovirus, the nonstructural protein S (NSs) is the RNA-silencing suppressor (RSS) and pathogenicity determinant. Here, we demonstrate that a putative α-helix, designated H8, spanning amino acids 338 to 369 of the C-terminal region of the NSs protein, is crucial for self-interaction of watermelon silver mottle virus NSs protein and that the H8 affects RSS function. Co-immunoprecipitation, yeast two-hybrid, and bimolecular fluorescence complementation analyses revealed that the triple point mutation (TPM) of H8 amino acids Y338A, H350A, and F353A resulted in NSs protein self-interaction dysfunction. Transient expression of H8-deleted (ΔH8) and TPM NSs proteins in Nicotiana benthamiana plants by agroinfitration indicated that these proteins have weaker RSS activity and are far less stable than wild-type (WT) NSs. However, an electrophoretic mobility assay revealed that small interfering RNA (siRNA) binding ability of TPM NSs protein is not compromised. The pathogenicity assay of WT NSs protein expressed by the attenuated turnip mosaic virus vector restored severe symptoms in recombinant-infected N. benthamiana plants but not for ΔH8 or TPM proteins. Taken together, we conclude that the H8 helix in the C-terminal region of NSs protein is crucial for stabilizing NSs protein through self-interaction to maintain normal functions of RSS and pathogenicity, but not for NSs-siRNA binding activity.

Early Detection of Tomato Spotted Wilt Virus by Hyperspectral Imaging and Outlier Removal Auxiliary Classifier Generative Adversarial Nets (OR-AC-GAN).

Tomato spotted wilt virus is a wide-spread plant disease in the world. It can threaten thousands of plants with a persistent and propagative manner. Early disease detection is expected to be able to control the disease spread, to facilitate management practice, and further to guarantee accompanying economic benefits. Hyperspectral imaging, a powerful remote sensing tool, has been widely applied in different science fields, especially in plant science domain. Rich spectral information makes disease detection possible before visible disease symptoms showing up. In the paper, a new hyperspectral analysis proximal sensing method based on generative adversarial nets (GAN) is proposed, named as outlier removal auxiliary classifier generative adversarial nets (OR-AC-GAN). It is an all-in-one method, which integrates the tasks of plant segmentation, spectrum classification and image classification. The model focuses on image pixels, which can effectively visualize potential plant disease positions, and keep experts' attention on these diseased pixels. Meanwhile, this new model can improve the performances of classic spectrum band selection methods, including the maximum variance principle component analysis (MVPCA), fast density-peak-based clustering, and similarity-based unsupervised band selection. Selecting spectrum wavebands reasonably is an important preprocessing step in spectroscopy/hyperspectral analysis applications, which can reduce the computation time for potential in-field applications, affect the prediction results and make the hyperspectral analysis results explainable. In the experiment, the hyperspectral reflectance imaging system covers the spectral range from 395 nm to 1005 nm. The proprosed model makes use of 83 bands to do the analysis. The plant level classification accuracy gets 96.25% before visible symptoms shows up. The pixel prediction false positive rate in healthy plants gets as low as 1.47%. Combining the OR-AC-GAN with three existing band selection algorithms, the performance of these band selection models can be significantly improved. Among them, MVPCA can leverage only 8 spectrum bands to get the same plant level classification accuracy as OR-AC-GAN, and the pixel prediction false positive rate in healthy plants is 1.57%, which is also comparable to OR-AC-GAN. This new model can be potentially transferred to other plant diseases detection applications. Its property to boost the performance of existing band selection methods can also accelerate the in-field applications of hyperspectral imaging technology.

Identification and localization of Tospovirus genus-wide conserved residues in 3D models of the nucleocapsid and the silencing suppressor proteins.

BACKGROUND: Tospoviruses (genus Tospovirus, family Peribunyaviridae, order Bunyavirales) cause significant losses to a wide range of agronomic and horticultural crops worldwide. Identification and characterization of specific sequences and motifs that are critical for virus infection and pathogenicity could provide useful insights and targets for engineering virus resistance that is potentially both broad spectrum and durable. Tomato spotted wilt virus (TSWV), the most prolific member of the group, was used to better understand the structure-function relationships of the nucleocapsid gene (N), and the silencing suppressor gene (NSs), coded by the TSWV small RNA. METHODS: Using a global collection of orthotospoviral sequences, several amino acids that were conserved across the genus and the potential location of these conserved amino acid motifs in these proteins was determined. We used state of the art 3D modeling algorithms, MULTICOM-CLUSTER, MULTICOM-CONSTRUCT, MULTICOM-NOVEL, I-TASSER, ROSETTA and CONFOLD to predict the secondary and tertiary structures of the N and the NSs proteins. RESULTS: We identified nine amino acid residues in the N protein among 31 known tospoviral species, and ten amino acid residues in NSs protein among 27 tospoviral species that were conserved across the genus. For the N protein, all three algorithms gave nearly identical tertiary models. While the conserved residues were distributed throughout the protein on a linear scale, at the tertiary level, three residues were consistently located in the coil in all the models. For NSs protein models, there was no agreement among the three algorithms. However, with respect to the localization of the conserved motifs, G18 was consistently located in coil, while H115 was localized in the coil in three models. CONCLUSIONS: This is the first report of predicting the 3D structure of any tospoviral NSs protein and revealed a consistent location for two of the ten conserved residues. The modelers used gave accurate prediction for N protein allowing the localization of the conserved residues. Results form the basis for further work on the structure-function relationships of tospoviral proteins and could be useful in developing novel virus control strategies targeting the conserved residues.

Distinct Mechanism for the Formation of the Ribonucleoprotein Complex of Tomato Spotted Wilt Virus.

The Tomato spotted wilt virus (TSWV) belongs to the Tospovirus genus of the Bunyaviridae family and represents the sole plant-infecting group within bunyavirus. TSWV encodes a nucleocapsid protein (N) which encapsidates the RNA genome to form a ribonucleoprotein complex (RNP). In addition, the N has multiple roles during the infection of plant cells. Here, we report the crystal structure of the full-length TSWV N. The N features a body domain consisting of an N-lobe and a C-lobe. These lobes clamp a positively charged groove which may constitute the RNA binding site. Furthermore, the body domains are flanked by N- and C-terminal arms which mediate homotypic interactions to the neighboring subunits, resulting in a ring-shaped N trimer. Interestingly, the C terminus of one protomer forms an additional interaction with the protomer of an adjacent trimer in the crystal, which may constitute a higher-order oligomerization contact. In this way, this study provides insights into the structure and trimeric assembly of TSWV N, which help to explain previous functional findings, but also suggests distinct N interactions within a higher-order RNP.IMPORTANCE TSWV is one of the most devastating plant pathogens that cause severe diseases in numerous agronomic and ornamental crops worldwide. TSWV is also the prototypic member of the Tospovirus genus, which is the sole group of plant-infecting viruses in the bunyavirus family. This study determined the structure of full-length TSWV N in an oligomeric state. The structural observations explain previously identified biological properties of TSWV N. Most importantly, the additional homotypic interaction between the C terminus of one protomer with another protomer indicates that there is a distinct mechanism of RNP formation in the bunyavirus family, thereby enhancing the current knowledge of negative-sense single-stranded RNA virus-encoded N. TSWV N is the last remaining representative N with an unknown structure in the bunyavirus family. Combined with previous studies, the structure of TSWV N helps to build a complete picture of the bunyavirus-encoded N family and reveals a close evolutionary relationship between orthobunyavirus, phlebovirus, hantavirus, and tospovirus.

Asymmetric Trimeric Ring Structure of the Nucleocapsid Protein of Tospovirus.

Tomato spotted wilt virus (TSWV), belonging to the genus Tospovirus of the family Bunyaviridae, causes significant economic damage to several vegetables and ornamental plants worldwide. Similar to those of all other negative-strand RNA viruses, the nucleocapsid (N) protein plays very important roles in its viral life cycle. N proteins protect genomic RNAs by encapsidation and form a viral ribonucleoprotein complex (vRNP) with some RNA-dependent RNA polymerases. Here we show the crystal structure of the N protein from TSWV. Protomers of TSWV N proteins consist of three parts: the N arm, C arm, and core domain. Unlike N proteins of other negative-strand RNA viruses, the TSWV N protein forms an asymmetric trimeric ring. To form the trimeric ring, the N and C arms of the N protein interact with the core domains of two adjacent N proteins. By solving the crystal structures of the TSWV N protein with nucleic acids, we showed that an inner cleft of the asymmetric trimeric ring is an RNA-binding site. These characteristics are similar to those of N proteins of other viruses of the family Bunyaviridae Based on these observations, we discuss possibilities of a TSWV encapsidation model.IMPORTANCE Tospoviruses cause significant crop losses throughout the world. Particularly, TSWV has an extremely wide host range (>1,000 plant species, including dicots and monocots), and worldwide losses are estimated to be in excess of $1 billion annually. Despite such importance, no proteins of tospoviruses have been elucidated so far. Among TSWV-encoded proteins, the N protein is required for assembling the viral genomic RNA into the viral ribonucleoprotein (vRNP), which is involved in various steps of the life cycle of these viruses, such as RNA replication, virus particle formation, and cell-to-cell movement. This study revealed the structure of the N protein, with or without nucleic acids, of TSWV as the first virus of the genus Tospovirus, so it completed our view of the N proteins of the family Bunyaviridae.

Induction of cell death by tospoviral protein NSs and the motif critical for cell death does not control RNA silencing suppression activity.

Groundnut bud necrosis virus induces necrotic symptoms in different hosts. Previous studies showed reactive oxygen species-mediated programmed cell death (PCD) resulted in necrotic symptoms. Transgenic expression of viral protein NSs mimics viral symptoms. Here, we showed a role for NSs in influencing oxidative burst in the cell, by analyzing H2O2 accumulation, activities of antioxidant enzymes and expression levels of vacuolar processing enzymes, H2O2-responsive microRNA 319a.2 plus its possible target metacaspase-8. The role of NSs in PCD, was shown using two NSs mutants: one in the Trp/GH3 motif (a homologue of pro-apototic domain) (NSsS189R) and the other in a non-Trp/GH3 motif (NSsL172R). Tobacco rattle virus (TRV) expressing NSsS189R enhanced the PCD response, but not TRV-NSsL172R, while RNA silencing suppression activity was lost in TRV-NSsL172R, but not in TRV-NSsS189R. Therefore, we propose dual roles of NSs in RNA silencing suppression and induction of cell death, controlled by different motifs.

Host-specific accumulation and temperature effects on the generation of dimeric viral RNA species derived from the S-RNA of members of the Tospovirus genus.

Polygonum ringspot virus (PolRSV) is a recently characterized Tospovirus reported in Italy. Northern blot analyses of PolRSV infections in Nicotiana benthamiana and tomato plants showed that a viral RNA species with nearly twice the length of the Small genomic RNA (S-RNA) accumulated abundantly in the former host, but was not detected in the latter. Additional assays confirmed that biogenesis of this novel RNA species was common to all PolRSV isolates tested and also to an isolate of Tomato spotted wilt virus (TSWV). Given its size, we hypothesized that the novel RNA species was a dimer molecule and we confirmed this hypothesis by RNA sequencing (RNAseq) analysis and reverse transcription (RT)-PCR of putative predicted dimer junction sites in RNA extracts of N. benthamiana challenged with PolRSV isolates Plg6 and Plg13/2. We also confirmed that these molecules are derived from head-to-tail dimers and often contain deletions at their junction sites. We named these novel molecules imperfect dimer RNAs (IMPD-RNAs). PolRSV IMPD-RNAs systemic accumulation in a range of host plants was restricted to N. benthamiana and Nicotiana occidentalis. Notably, IMPD-RNAs accumulation was modulated by temperature and their generation was restricted to late stages of systemic infection (12 days post-inoculation) in N. benthamiana. Differently from all other PolRSV isolates used in this study, Plg13/2 generated more IMPD-RNAs coupled with low amounts of genomic S-RNA and maintained them even at 18 °C, besides having lost the ability to infect tomato plants. This is the first characterization of S-RNA dimers for Tospovirus, and of occurrence of dimers of genomic segments at the whole organism level for Bunyaviridae.

The complete genome of the tospovirus Zucchini lethal chlorosis virus.

BACKGROUND: Zucchini lethal chlorosis virus (ZLCV) causes significant losses in the production of cucurbits in Brazil. This virus belongs to the genus Tospovirus (family Bunyaviridae) and seems to be exclusively transmitted by Frankliniella zucchini (Thysanoptera). Tospoviruses have a tripartite and single-stranded RNA genome classified as S (Small), M (Medium) and L (Large) RNAS. Although ZLCV was identified as a member of the genus Tospovirus in 1999, its complete genome had not been sequenced until now. FINDINGS: We sequenced the full-length genome of two ZLCV isolates named ZLCV-SP and ZLCV-DF. The phylogenetic analysis showed that ZLCV-SP and ZLCV-DF clustered with the previously reported isolate ZLCV-BR09. Their proteins were closely related, except the non-structural protein (NSm), which was highly divergent (approximately 90 % identity). All viral proteins clustered similarly in our phylogenetic analysis, excluding that these ZLCV isolates have originated from reassortment events of different tospovirus species. CONCLUSION: Here we report for the first time the complete genome of two ZLCV isolates that were found in the field infecting zucchini and cucumber.

Comparison of small RNA profiles in Nicotiana benthamiana and Solanum lycopersicum infected by polygonum ringspot tospovirus reveals host-specific responses to viral infection.

Viral small RNAs (vsRNAs) are one of the key elements involved in RNA silencing-based defense against viruses in plants. We analyzed the vsRNA profiles in Nicotiana benthamiana and Solanum lycopersicum infected by polygonum ringspot virus (PolRSV) (Tospovirus, Bunyaviridae). VsRNAs were abundant in both hosts, but a different size profile was observed, with an abundance peak at 21 in N. benthamiana and at 22 nt in tomato. VsRNAs mapping to the PolRSV L genomic segment were under-represented in both hosts, while S and M segments were differentially and highly targeted in N. benthamiana and tomato, respectively. Differences in preferential targeting of single ORFs were observed, with over-representation of NSs ORF-derived reads in N. benthamiana. Intergenic regions (IGRs)-mapping vsRNAs were under-represented, while enrichment of vsRNAs reads mapping to the NSs positive sense strand was observed in both hosts. Comparison with a previous study on tomato spotted wilt virus (TSWV) under the same experimental conditions, showed that the relative accumulation of PolRSV-specific and endogenous sRNAs was similar to the one observed for silencing suppressor-deficient TSWV strains, suggesting possible different properties of PolRSV NSs silencing suppressor compared to that of TSWV.

Homology modeling and molecular dynamics provide structural insights into tospovirus nucleoprotein.

BACKGROUND: Tospovirus is a plant-infecting genus within the family Bunyaviridae, which also includes four animal-infecting genera: Hantavirus, Nairovirus, Phlebovirus and Orthobunyavirus. Compared to these members, the structures of Tospovirus proteins still are poorly understood. Despite multiple studies have attempted to identify candidate N protein regions involved in RNA binding and protein multimerization for tospovirus using yeast two-hybrid systems (Y2HS) and site-directed mutagenesis, the tospovirus ribonucleocapsids (RNPs) remains largely uncharacterized at the molecular level and the lack of structural information prevents detailed insight into these interactions. RESULTS: Here we used the nucleoprotein structure of LACV (La Crosse virus-Orthobunyavirus) and molecular dynamics simulations to access the structure and dynamics of the nucleoprotein from tospovirus GRSV (Groundnut ringspot virus). The resulting model is a monomer composed by a flexible N-terminal and C-terminal arms and a globular domain with a positively charged groove in which RNA is deeply encompassed. This model allowed identifying the candidate amino acids residues involved in RNA interaction and N-N multimerization. Moreover, most residues predicted to be involved in these interactions are highly conserved among tospoviruses. CONCLUSIONS: Crucially, the interaction model proposed here for GRSV N is further corroborated by the all available mutational studies on TSWV (Tomato spotted wilt virus) N, so far. Our data will help designing further and more accurate mutational and functional studies of tospovirus N proteins. In addition, the proposed model may shed light on the mechanisms of RNP shaping and could allow the identification of essential amino acid residues as potential targets for tospovirus control strategies.

Complete Genome Sequence of Mulberry Vein Banding Associated Virus, a New Tospovirus Infecting Mulberry.

Mulberry vein banding associated virus (MVBaV) that infects mulberry plants with typical vein banding symptoms had been identified as a tentative species of the genus Tospovirus based on the homology of N gene sequence to those of tospoviruses. In this study, the complete sequence of the tripartite RNA genome of MVBaV was determined and analyzed. The L RNA has 8905 nucleotides (nt) and encodes the putative RNA-dependent RNA polymerase (RdRp) of 2877 aa amino acids (aa) in the viral complementary (vc) strand. The RdRp of MVBaV shares the highest aa sequence identity (85.9%) with that of Watermelon silver mottle virus (WSMoV), and contains conserved motifs shared with those of the species of the genus Tospovirus. The M RNA contains 4731 nt and codes in ambisense arrangement for the NSm protein of 309 aa in the sense strand and the Gn/Gc glycoprotein precursor (GP) of 1,124 aa in the vc strand. The NSm and GP of MVBaV share the highest aa sequence identities with those of Capsicum chlorosis virus (CaCV) and Groundnut bud necrosis virus (GBNV) (83.2% and 84.3%, respectively). The S RNA is 3294 nt in length and contains two open reading frames (ORFs) in an ambisense coding strategy, encoding a 439-aa non-structural protein (NSs) and the 277-aa nucleocapsid protein (N), respectively. The NSs and N also share the highest aa sequence identity (71.1% and 74.4%, respectively) with those of CaCV. Phylogenetic analysis of the RdRp, NSm, GP, NSs, and N proteins showed that MVBaV is most closely related to CaCV and GBNV and that these proteins cluster with those of the WSMoV serogroup, and that MVBaV seems to be a species bridging the two subgroups within the WSMoV serogroup of tospoviruses in evolutionary aspect, suggesting that MVBaV represents a distinct tospovirus. Analysis of S RNA sequence uncovered the highly conserved 5'-/3'-ends and the coding regions, and the variable region of IGR with divergent patterns among MVBaV isolates.

Demonstration of helicase activity in the nonstructural protein, NSs, of the negative-sense RNA virus, groundnut bud necrosis virus.

The nonstructural protein NSs, encoded by the S RNA of groundnut bud necrosis virus (GBNV) (genus Tospovirus, family Bunyaviridae) has earlier been shown to possess nucleic-acid-stimulated NTPase and 5' α phosphatase activity. ATP hydrolysis is an essential function of a true helicase. Therefore, NSs was tested for DNA helicase activity. The results demonstrated that GBNV NSs possesses bidirectional DNA helicase activity. An alanine mutation in the Walker A motif (K189A rNSs) decreased DNA helicase activity substantially, whereas a mutation in the Walker B motif resulted in a marginal decrease in this activity. The parallel loss of the helicase and ATPase activity in the K189A mutant confirms that NSs acts as a non-canonical DNA helicase. Furthermore, both the wild-type and K189A NSs could function as RNA silencing suppressors, demonstrating that the suppressor activity of NSs is independent of its helicase or ATPase activity. This is the first report of a true helicase from a negative-sense RNA virus.

Tospovirus ambisense genomic RNA segments use almost complete repertoire of stable tetraloops in the intergenic region.

The intergenic regions of the ambisense RNA segments of viruses from the Tospovirus genus form large extended RNA structures that regulate virus replication. Using comparative structure analysis, we show the presence of conserved alternative conformations at the apical parts of these structures. In one conformation, a branched Y-shape, the 5'-proximal hairpin arms are mostly capped by exceptionally stable tetraloop motifs. The tetraloop hairpins are folded in both virus and virus-complementary sense RNAs, and different tetraloops can functionally replace each other. Folding simulations show that the branched Y-shape structures can undergo a conformational transition to alternative extended rod-like conformations. Functional importance of both alternatives is supported by nucleotide covariations. The balanced equilibrium between alternative structures is evidenced by native gel electrophoresis of mutant RNA transcripts with shifted equilibria. The tetraloops play a role in the stability and dynamics of structures but may also be recognized by proteins involved in translation and/or replication.

Diverging affinity of tospovirus RNA silencing suppressor proteins, NSs, for various RNA duplex molecules.

The tospovirus NSs protein was previously shown to suppress the antiviral RNA silencing mechanism in plants. Here the biochemical analysis of NSs proteins from different tospoviruses, using purified NSs or NSs containing cell extracts, is described. The results showed that all tospoviral NSs proteins analyzed exhibited affinity to small double-stranded RNA molecules, i.e., small interfering RNAs (siRNAs) and micro-RNA (miRNA)/miRNA* duplexes. Interestingly, the NSs proteins from tomato spotted wilt virus (TSWV), impatiens necrotic spot virus (INSV), and groundnut ringspot virus (GRSV) also showed affinity to long double-stranded RNA (dsRNA), whereas tomato yellow ring virus (TYRV) NSs did not. The TSWV NSs protein was shown to be capable of inhibiting Dicer-mediated cleavage of long dsRNA in vitro. In addition, it suppressed the accumulation of green fluorescent protein (GFP)-specific siRNAs during coinfiltration with an inverted-repeat-GFP RNA construct in Nicotiana benthamiana. In vivo interference of TSWV NSs in the miRNA pathway was shown by suppression of an enhanced GFP (eGFP) miRNA sensor construct. The ability to stabilize miRNA/miRNA* by different tospovirus NSs proteins in vivo was demonstrated by increased accumulation and detection of both miRNA171c and miRNA171c* in tospovirus-infected N. benthamiana. All together, these data suggest that tospoviruses interfere in the RNA silencing pathway by sequestering siRNA and miRNA/miRNA* molecules before they are uploaded into their respective RNA-induced silencing complexes. The observed affinity to long dsRNA for only a subset of the tospoviruses studied is discussed in light of evolutional divergence and their ancestral relation to the animal-infecting members of the Bunyaviridae.

The cytoplasmic domain of tomato spotted wilt virus Gn glycoprotein is required for Golgi localisation and interaction with Gc.

Envelopment of tomato spotted wilt virus nucleocapsids occurs at the Golgi stacks of infected cells. This is also the place where the two membrane glycoproteins Gn and Gc accumulate upon coexpression. The required Golgi retention signal has previously been demonstrated to reside within Gn. Using a series of truncated Gn proteins, the Golgi retention signal was mapped to a stretch of 10 amino acids on this protein's cytoplasmic tail, 20 residues downstream the transmembrane domain. Studies on the intracellular distribution of chimeric Gc proteins in which the cytoplasmic tail and/or transmembrane domain were exchanged by those from Gn, demonstrated the additional requirement of the Gn transmembrane domain for Golgi targeting. Truncated Gn constructs lacking the C-terminal 20 amino acids but still localising to the Golgi were no longer able to redirect Gc, suggesting the requirement of this domain for interaction with Gc.

Tomato spotted wilt virus Gc and N proteins interact in vivo.

Tomato spotted wilt virus (TSWV) virions consist of a nucleocapsid core surrounded by a membrane containing glycoproteins Gn and Gc. To unravel the protein interactions involved in the membrane acquisition of RNPs, TSWV nucleocapsid protein (N), Gn and Gc were expressed and analyzed in BHK21 cells. Upon coexpression of Gn, Gc and N, a partial colocalization of N with both glycoproteins was observed in the Golgi region. In contrast, upon coexpression of Gc and N in the absence of Gn, both proteins colocalized to a distinct non-Golgi perinuclear region. Using FLIM and FRET, interaction was demonstrated between N and Gc, but not between N and Gn, and was only observed in the region where both proteins accumulated. The genuine character of N-Gc interaction was confirmed by its presence in purified virus and RNP preparations. The results are discussed in view of TSWV particle assembly taking place at the Golgi complex.

Immunodiagnosis of groundnut and watermelon bud necrosis viruses using polyclonal antiserum to recombinant nucleocapsid protein of Groundnut bud necrosis virus.

In vitro gene expression strategy was used for the production of polyclonal antiserum to the nucleocapsid protein (NP) of Groundnut bud necrosis virus (GBNV). The GBNV NP gene from cowpea isolate was cloned into 6x His-tagged UA cloning vector and expressed in Escherichia coli [M15] cells. The fusion protein was detected in insoluble fraction and was purified by using Ni-NTA agarose resin. The purified 6x His-fusion protein ( approximately 32 kDa) was used for immunisation to produce a high titre polyclonal antiserum. The antiserum to the NP of GBNV at 1:4000 dilution detected successfully natural infection of GBNV and Watermelon bud necrosis virus in a wide range of cucurbitaceous, leguminous and solanaceous hosts from different locations.

Purification and serological analyses of tospoviral nucleocapsid proteins expressed by Zucchini yellow mosaic virus vector in squash.

A plant viral vector engineered from an in vivo infectious clone of Zucchini yellow mosaic virus (ZYMV) was used to express the nucleocapsid proteins (NPs) of tospoviruses in planta. The open reading frames (ORFs) of NPs of different serogroups of tospoviruses, including Tomato spotted wilt virus, Impatiens necrotic spot virus, Watermelon silver mottle virus, Peanut bud necrosis virus, and Watermelon bud necrosis virus (WBNV), were in frame inserted in between the P1 and HC-Pro genes of the ZYMV vector. Six histidine residues and an NIa protease cleavage site were added at the C-terminal region of the inserts to facilitate purification and process of free form of the expressed NPs, respectively. Approximately 1.2-2.5 mg/NPs 100 g tissues were purified from leaf extracts of zucchini squash. The expressed WBNV NP was used as an immunogen for the production of highly specific polyclonal antisera and monoclonal antibodies. The procedure provides a convenient and fast way for production of large quantities of pure NPs of tospoviruses in planta. The system also has a potential for production of any proteins of interest in cucurbits.